| Total glucosides from Radix gentianae. (TGG), which was extracted and purified from Gentiana scabra Bge. shows a wide range of physiological activity on the basis of modern pharmacological researches, such as liver protection and promoting bile secretion, promoting digestion, anti-imflammation, antibacterial and influence on central nervous system. If TGG is prepared into injection with modern technology, its curative effect must be enhanced and adapt to treating acute and heavy disease such as hepatitis. The purpose of this project is to extract and purify TGG from Gentiana scabra Bge., prepare the lyophilized powder, establish its pharmaceutical qualities and investigate its pharmacokinetics. In this project, we focus on increasing the percentage of the active ingredients (TGG) in extract, reduce the content of the impurity, improve the antiviral curative and reduce its immediate hypersensitivity and the side effect.1. Study on the resources and quality of Gentiana scabra Bge.(1) A survey of main planting area of Gentiana scabra Bge.. The quality of 21 batch of Gentiana scabra Bge. from different origin were compared and main sources of raw material were chosed.(2) The quality standards of Gentiana scabra Bge.was established, which mainly includes fingerprint and determination of TGG, gentiopicrin, sweroside and swertiamarian. Samples from different habitats were of high similarity, though a few samples showed evident difference in fingerprint chromatograms. The results of similarity analisis for chromatographic fingerprint were basically consistent with those of quantitative analysis.2. Optimization of extraction and purification process for effective ingredients (TGG)(1) Optimization of extraction process for effective ingredients in Gentiana scabra Bge.. The extraction process was investigated by soaking, transonic, hot refluxing, percolation and decoction with water. The extraction technique of Gentiana scabra Bge. was optimized by using orthogonal test design and the synthetic score based on the extraction rate of gentiopicrin, TGG and content of gentiopicrin in total solid was used as the assessment indices.(2) TGG were purified from Gentiana scabra Bge. with macroreticular resin. Purification is the key step to guarantee the quality of TGG extract. The adsorption capacity, elutive ratio and purity of gentiopicrin in the product were used as indexes to select the best resin. The technical procedure for purification of gentiopicrin with the optimum macroreticular resin was screened by content and transferring rate of gentiopicrin in product. A central composite design-response surface methodology was also employed to study the technological parameters of the purification process with HPD-300 type macroreticular resin. Similarity of 10 batches of TGG was more than 0.98, which showed the preparation process was stable and repeatable.3. Study on stability of TGG(1) Stability of gentiopicrin in biological fluids in vitro. In vitro stability of gentiopicrin in phosphate buffer solutions with different pH, gastrointestinal contents and mucosa homogenates, liver homogenate and plasma were investigated and the main possible site for metabolism and the factors which affect the stability of gentiopicrin were revealed.(2) The aqueous stability of gentiopicrin was studied. The effect of temperature, pH, buffer species, buffer concentration, ionic strength and light on the stability of gentiopicrin solution were investigated and the most appropriate preservation conditions for TGG were determined.4. Formulation and preparation of lyophilized injection of TGGAs TGG is unstable in water, easily damaged in gastrointestinal tract and of low oral bioavailability, so lyophilized injection was chosen as its dosage form. The formulation of TGG lyophilization was optimized based on its texture, color and resolubility. The final product was yellowish freeze-dried powder and solved easily in water.Compatibility of lyophilized TGG with five kinds of infusion solutions was also evaluated at room temperature for 12 h. The stability of the mixtures was characterized in respect of appearance, clarity, pH and content of effective ingredients.5. Quality standards and stability of lyophilization of TGGA strict quality control of lyophilized injection of TGG was established, which includs characters, identification, inspection (gross deviation, pH, moisture , visible particles, clarity, tannin, resin, oxalate, protein, residue of burn, potassium ion, heavy metal), fingerprint and determination of TGG, gentiopicrin, sweroside and swertiamarian. 3 batches of the product were examined according to the standard, the results showed that the process was stable, and meet the requirement of quality standard.The stability of of lyophilized injection of TGG was investigated under accelerated conditions and room temperature by taking characters, moisture, fingerprint and content as criterions.6. Investigation on pharmacokinetics of gentiopicrin in Beagle dogsA RP-HPLC method for the determination of gentiopicrin in plasma was established to investigate its pharmacokinetic characteristics in beagle dogs.The pharmacokinetics of gentiopicrin after i.v. and p.o. administration showed a rapid absorption, distribution and elimination process in Beagle dogs. The absolute bioavailability of gentiopicrin given orally was ratherly low. |
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